Relationship between Indel Variants within the JAK2 Gene and Growth Traits in Goats

Simple Summary Janus kinase 2 (JAK2) plays a critical role in myoblast proliferation and fat deposition in animals, and our previous RNA-Seq analyses suggest that it is a candidate gene closely linked to muscle development. The effect of this gene on the body characteristics of animals has been investigated to a limited extent. Here, an analysis of 548 samples from three breeds examined the relationship between two insertion/deletion polymorphisms (del19008 and del72416) of the goat JAK2 gene and body traits. The results showed that del19008 was associated with body height, body length, and trunk index. Locus del72416 was associated with BL, BH, TI, HuW, ChC, ChW, BLI, ChCI, CWI, and HuWI. In addition, the effect of the two InDels on the Nubian group was emphasized in terms of body height. Thus, these loci should be able to provide molecular markers for improving the body traits of goats. Abstract Janus kinase 2 (JAK2) plays a critical role in myoblast proliferation and fat deposition in animals. Our previous RNA-Seq analyses identified a close association between the JAK2 gene and muscle development. To date, research delving into the relationship between the JAK2 gene and growth traits has been sparse. In this study, we sought to investigate the relationship between novel mutations within the JAK2 gene and goat growth traits. Herein, two novel InDel (Insertion/Deletion) polymorphisms within the JAK2 gene were detected in 548 goats, and only two genotypes were designated as ID (Insertion/Deletion) and DD (Deletion/Deletion). The results indicate that the two InDels, the del19008 locus in intron 2 and del72416 InDel in intron 6, showed significant associations with growth traits (p < 0.05). Compared to Nubian and Jianzhou Daer goats, the del72416 locus displayed a more pronounced effect in the Fuqing breed group. In the Nubian breed (NB) group, both InDels showed a marked influence on body height (BH). There were strong linkages observed for these two InDels between the Fuqing (FQ) and Jianzhou (JZ) populations. The DD-ID diplotype was associated with inferior growth traits in chest width (ChW) and cannon circumference (CaC) in the FQ goats compared to the other diplotypes. In the NB population, the DD-DD diplotype exhibited a marked negative impact on BH and HuWI (hucklebone width index), in contrast to the other diplotypes. In summary, our findings suggest that the two InDel polymorphisms within the JAK2 gene could serve as valuable molecular markers for enhancing goat growth traits in breeding programs.


Introduction
The Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway is an important intracellular signal transduction pathway [1].Many JAK/STAT receptor agonists, such as growth hormone, prolactin, and gamma-interferon, send signals more directly to the nucleus than receptor tyrosine kinases [2,3].Through in-depth research on the JAK2/STAT3 signaling pathway, it has been discovered that numerous cytokines (e.g., IFN, IL-2, IL-4, IL-6, and CNTF) and growth factors (e.g., EGF, PDGF, and CSF) utilize this pathway to induce cell proliferation, differentiation, or apoptosis [4].Jak2 is typically activated by binding to type I or type II cytokine receptors and their corresponding ligands; it then phosphorylates STAT3, promoting its translocation to the nucleus and ultimately inducing the expression of target genes [5].They play both specific and pleiotropic biological functions in embryonic development, immune function regulation, hematopoietic cell generation, and tumor occurrence [6].This indicates the pivotal regulatory function of JAK2/STAT3 signaling in cellular processes [7].
Janus kinase (JAK) is a protein tyrosine kinase that plays an important role in the cytokine signaling pathway [8] and has four members, namely, JAK1, JAK2, JAK3, and TYK2.Jak2 plays a critical role in the signaling pathways of numerous cytokine receptors [9,10], which is a key transcription factor for myoblast proliferation and fat deposition [11,12].A report showed that JAK2 is a GH receptor that undergoes ligand-dependent nuclear translocation and phosphorylates upon ligand binding with the GH receptor [13].The disruption of hepatocyte growth GH signaling via the disruption of jak2 (JAK2L) leads to a fatty liver [14].Following the administration of the jak inhibitor AG490 and transfection with JAK2-siRNA, there was a decline in the levels of MHC, MGN, MEF2, and MyoD.Additionally, the introduction of JAK2-siRNA markedly lowered the luciferase reporter gene activity that is dependent on myogenin-, MRF-, MEF2-, and MyoD.The expression of the IGF2 gene and the HGF gene is also regulated by the JAK2 gene [15].
Jak2 can influence the development of skeletal muscle and energy metabolism in organisms [16].The JAK2 gene was screened as a key candidate gene related to muscle growth and development in our previous RNA sequence [17].It has been found that the JAK2 gene is involved in the metabolism of body fat and that a decrease in the expression of this pathway leads to obesity [18].Reports have revealed that mice with a liver-specific disruption of the GH signaling mediator JAK2 develop severe fatty livers and reduced body fat in mouse experiments [19].JAK2 deficiency can lead to ischemia and the death of the embryo due to the failure of normal erythropoiesis in mice [20], and its abnormal activation also leads to the occurrence of blood diseases such as leukemia [21].In cattle, JAK2 is related to lactation function, and it has been found that, when the expression of JAK2 rises, the activity of the JAK2-STAT signaling pathway increases, which can improve the lactation performance of dairy cows, such as milk protein production.Previous studies have demonstrated an association between the JAK2 gene and growth traits in sheep [22] and cattle [23].Hitherto, the JAK2 gene has been considered a possible target for improving animal growth traits.As one of the many identified mutations, InDel is high in density and readily genotyped by fragment length polymorphisms.Genome-wide InDel polymorphisms among different accessions can be detected using whole-genome resequencing to guide the development of molecular markers.However, there have been no documented instances of InDel polymorphisms in the JAK2 gene of goats.This study aimed to further detect the association between the novel InDel mutation within the goat JAK2 gene and growth traits.In total, 548 samples from three breeds were used in this research, which may be useful for marker-assisted selection (MAS) in goat breeding.

Materials and Methods
The protocols (protocol number: 202207FJ002) of the Faculty of Animal Policy and Welfare Committee of the Fujian Academy of Agricultural Sciences (FAAS) for the use and care of animals in research were followed throughout all the experimental procedures.

DNA Separation and Creation of Genomic DNA Pools
Total DNA was isolated from the blood of 548 individuals using phenol-chloroform extraction, as described in Reference [24].The DNA obtained was then diluted to a concentration of 50 ng/µL and stored at −80 degrees Celsius to facilitate the construction of genomic pools and enable polymorphism identification.In constructing the DNA pool, we randomly selected 50 samples for each breed, which were used to detect insertion-deletion mutations within the JAK2 gene.

Primer Design, PCR Amplifications, and Genotyping
The sequence data and variation details of the JAK2 gene (NC_030815.1)were obtained from the NCBI (Accessed on 7 July 2023.https://www.ncbi.nlm.nih.gov/) and Ensembl databases (Accessed on 8 July 2023.http://asia.ensembl.org/index.html).The primer pair for predicted mutation locus detecting was designed by the NCBI online primer design software (Accessed on 8 July 2023.https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=Blast Home Primer) (Table 1).The DNA mixed pool was used to detect the novel InDel locus of the JAK2 gene.The PCR products were sequenced by Sangon Biotec Co., Ltd.(Shanghai, China).Each PCR reaction was performed in a 25 µL reaction mixture containing 50 ng genomic DNA; 0.5 µM of each primer; 1× buffer (including 1.5 mM MgCl 2 ); 200 µM of the dNTPs (dATP, dTTP, dCTP, and dGTP); and 0.625 units of Taq DNA polymerase (TaKaRa, Dalian, China).The cycling protocol used in our previous study was followed [26].Eventually, PCR products were detected via electrophoresis with 3.5% agarose gel stained with ethidium bromide at 120 V for 50 min.Two InDel variants were detected in the goat JAK2 gene using primer pairs P4 and P13, namely, "del19008" and "del72416", respectively.

Bioinformatics Analysis
The alignment of nucleotide and protein sequences was carried out with the assistance of MEGA software version 5.1 (Accessed on 20 July 2023.available at http://www.megasoftware.net/)and BioXM 2.6 (Nanjing Agricultural University, Nanjing, China).Evolutionary trees were generated through the neighbor-joining algorithm, as facilitated by MEGA version 5.1 and NCBI pairwise alignments (Accessed on 20 July 2023.http://www.ncbi.nlm.nih.gov/blast).

Statistical Analyses
We used the GDIcall online calculator (Accessed on 23 August 2023.http://www.msrcall.com/Gdicall.aspx)to calculate the genotypic and allelic frequencies of goat InDels in the JAK2 gene and to examine the polymorphism information content (PIC).Hardy-Weinberg equilibrium (HWE) was tested using the χ 2 test.Since only two genotypes were observed in each variant, an independent-sample t-test was used to analyze the correlation between the InDel and growth traits.The association between combined genotypes and growth traits was analyzed through ANOVA.SPSS statistical software (version 18.0) (IBM Corp., Armonk, NY, USA) was used for the calculations.Y ijk = µ + α i + β j + H k + ε ijk was designed to investigate the association of the two InDels with growth traits, where Y ijk was the observation of the growth trait (body height, etc.) evaluated on the ith level of the fixed factor age (α i ) and the jth level of the fixed factor genotype (β j ); µ was the overall mean for each growth trait; H k was the fixed effect of the hatch; and ε ijk was the random error for the ijkth individual [26].

Genotypic and Genetic Parameters of InDels within the Goat JAK2 Gene
Two novel deletion polymorphisms, del19008 and del72416, were detected in the JAK2 gene using DNA pool sequencing, based on the predicted sequences from the European Variation Archive database (Accessed on 8 July 2023.http://www.ebi.ac.uk/eva/,Variant IDs: rs664724553 and rs670284366, respectively).Specifically, the del19008 locus is situated within the second intron of the JAK2 gene on chromosome 8 of the goat (NC_030815.1:g.19008_19013delTCCAT), while the del72416 locus is found in the sixth intron of the gene (NC_030815.1:g.72416_72426delCTGTACGGTA).DNA gel electrophoresis revealed only two genotypes at these loci: DD (deletion/deletion) and ID (insertion/deletion), as shown in Figure 1.

Linkage Disequilibrium (LD) and Combined Genotype Analysis
The linkage disequilibrium analysis of the loci del19008 and del72416 was conducted using Haploview 4.2 software (Accessed on 15 September 2023.http://www.broad.mit.edu/mpg/haploview/), in which D (0 < D < 1) and r 2 (0 < r 2 < 1) are indicators used to measure the strength of association across two linked genetic loci.

Genotypic and Genetic Parameters of InDels within the Goat JAK2 Gene
Two novel deletion polymorphisms, del19008 and del72416, were detected in the JAK2 gene using DNA pool sequencing, based on the predicted sequences from the European Variation Archive database (Accessed on 8 July 2023.http://www.ebi.ac.uk/eva/,Variant IDs: rs664724553 and rs670284366, respectively).Specifically, the del19008 locus is situated within the second intron of the JAK2 gene on chromosome 8 of the goat (NC_030815.1:g.19008_19013delTCCAT), while the del72416 locus is found in the sixth intron of the gene (NC_030815.1:g.72416_72426delCTGTACGGTA).DNA gel electrophoresis revealed only two genotypes at these loci: DD (deletion/deletion) and ID (insertion/deletion), as shown in Figure 1.

Genetic Diversity of the Two InDels within the JAK2 Gene in Three Goat Populations
The genotypic and allelic frequencies, as well as polymorphism measures, were calculated based on genotype data (Table 2), including gene homozygosity (Ho), heterozygosity (He), effective allele numbers (Ne), and polymorphism information content (PIC).The chi-square (χ 2 ) test was employed to assess whether the polymorphisms at loci del19008 and del72416 conformed to Hardy-Weinberg equilibrium (HWE).The results revealed that, in the three goat populations, the frequency of the ID genotype was higher

Genetic Diversity of the Two InDels within the JAK2 Gene in Three Goat Populations
The genotypic and allelic frequencies, as well as polymorphism measures, were calculated based on genotype data (Table 2), including gene homozygosity (Ho), heterozygosity (He), effective allele numbers (Ne), and polymorphism information content (PIC).The chi-square (χ 2 ) test was employed to assess whether the polymorphisms at loci del19008 and del72416 conformed to Hardy-Weinberg equilibrium (HWE).The results revealed that, in the three goat populations, the frequency of the ID genotype was higher than that of the DD genotype for both loci.Accordingly, the frequency of the "D" allele was greater than that of the "I" allele.The genetic diversity at both loci was moderate (0.25 < PIC < 0.5) in the tested populations, as indicated by their PIC values (Table 2).The χ 2 test results indicated that neither of the two mutation loci was in HWE in the three populations (p < 0.05).

Bioinformatics Analysis of JAK2
The goat JAK2 mRNA sequence was found to encode a protein consisting of 1132 amino acids.We observed that the coding sequence of goat JAK2 shares 99%, 99%, 99%, 99%, 99%, 96%, 93%, 94%, 96%, and 94% similarity with Ovis aries (XP_042099355.than that of the DD genotype for both loci.Accordingly, the frequency of the "D" allele was greater than that of the "I" allele.The genetic diversity at both loci was moderate (0.25 < PIC < 0.5) in the tested populations, as indicated by their PIC values (Table 2).The χ 2 test results indicated that neither of the two mutation loci was in HWE in the three populations (p < 0.05).

Association between InDel Variants and Growth Traits in Goats
The analysis of the association between the InDel locus and phenotypic growth characteristics revealed a significant correlation with several growth traits in goats (p < 0.05) (Table 3).For the del19008 locus, individuals with ID genotypes displayed a more

Association between InDel Variants and Growth Traits in Goats
The analysis of the association between the InDel locus and phenotypic growth characteristics revealed a significant correlation with several growth traits in goats (p < 0.05) (Table 3).For the del19008 locus, individuals with ID genotypes displayed a more superior growth trait than DD with BH (p = 0.014) and TI (p = 0.009) in the group of NB and with HuW (p = 0.013) in the FQ breed, as well as CWI (p = 0.019) in the JZ population.The del72416 locus was highly associated with growth traits, including BL (p = 0.019), HuW (p = 0.011), ChC (p = 0.016), and ChW (p = 0.002) in the FQ goats and BH (p = 0.004), BLI (p = 0.00004), ChCI (p = 0.017), CWI (p = 0.046), and HuWI (p = 0.004) of the NB breed.In summary, the ID genotype was associated with more favorable growth traits at the del19008 locus, whereas, at the del72416 locus, the DD genotype was associated with better growth traits than the ID genotype.

LD and Haplotype Analysis
In the FQ and JZ populations, a strong LD was found between the loci del19008 and del72416, as indicated by the correlation coefficients (FQ: D' = 1.000, r 2 = 0.518; JZ: D' = 1.000, r 2 = 0.326), while moderate LD was observed in the NB population (D' = 0.333, r 2 = 0.108) (Figure 3).Subsequently, a haplotype association analysis was conducted with a minimum frequency criterion of 0.001 for haplotype consideration (Table 4).Among the four identified haplotypes, the ID-ID genotype exhibited the highest frequency in both the FQ and NB populations.Correlation analyses of the four combined genotypes in the FQ and NB goats indicated that the DD-ID diplotype was associated with significantly inferior growth traits than the other haplotypes in ChW (p = 0.003) and CaC (p = 0.011) in FQ goats.In the NB population, the DD-DD diplotype showed extremely significant inferiority in BH (p = 0.001) and HuWI (p = 6 × 10 −7 ) compared to the other diplotypes.Conversely, the DD-DD diplotype was associated with extremely significant superiority in the BLI (p = 1 × 10 −6 ) and ChCI (p = 6 × 10 −7 ) compared to the other diplotypes.These findings provide further evidence for the significant role of the JAK2 gene in influencing growth traits in both FQ and NB goat breeds.or stat5 elevates the circulating GH levels, which could exacerbate GH-mediated processes such as lipolysis in adipose tissue [14].Moreover, an expanding body of research is exploring the molecular mechanisms of jak2 function in muscle tissues [33,34].In particular, studies have found that the JAK2/STAT2/STAT3 pathway regulates the expression of HGF and IGF2, which are crucial for myoblast proliferation and differentiation [35].Jak2 is essential for development, and its conservation across mammals has been established [35,36].Conventional knockout mouse models show postnatal or embryonic lethality [8], possibly explaining the absence of homozygous deletions for the two InDels in the three goat populations.A protein similarity analysis indicated that goat JAK2 shares over 94% similarity with ten other species, highlighting its high conservation in ruminants.Screening results for InDel loci revealed only two out of seventeen predicted loci (Table 1), further indicating the conservative nature of the JAK2 gene.These findings underscore the vital role of JAK2 in the developmental processes of goats.
The genotype frequencies of the two InDels were found to be similar among the FQ, NB, and JZ goats (Table 2).The two InDels exhibited two genotypes: homozygous wild-type (DD) and heterozygous genotype (ID).The "D" allele was the predominant allele in all three goat breeds.The absence of the homozygous mutant genotype (II) was evident in the results of the HWE test.Moreover, a significant departure from HWE was noted in these breeds, likely due to non-random mating resulting from population stratification, selection, or genetic drift [37].The two InDels showed strong linkage in the FQ and JZ populations and moderate linkage disequilibrium in the NB group.The Fuqing goat is a distinct local breed from Fujian Province, China [38], while the Nubian goat originates from Egypt.The Jianzhou Daer goat is a cross between local Jianzhou and Nubian goats [39].It can be hypothesized that recombination between del19008 and del72416 occurred during the evolutionary selection process in these breeds.
In molecular breeding, the pivotal economic traits of growth and body weight performance are of increasing importance.Jak2 is an integral part of the growth hormone receptor pathway, involved in transmitting signals from class II cytokine receptors [5], and plays a critical role in the growth, development, and reproductive traits of ruminants [23].Previous research has mostly concentrated on the impact of SNPs within the JAK2 gene on cattle and sheep traits, such as growth [40], milk production [41], and mastitis resistance [42].However, the association between InDel polymorphisms of the JAK2 gene and growth traits in goats has been less investigated.This study aimed to address this gap by examining the association of two InDel polymorphisms of the JAK2 gene with growth traits in goats.The del19008 polymorphism was found to primarily affect the BH, HuW, and other traits in both FQ and NB breeds (Table 4), whereas the del72416 polymorphism mainly impacted the BH, BL, HuW, ChC, ChW, and other traits in the same breeds.Nevertheless, these InDels did not have a significant effect on the JZ group.The del72416 locus's influence was more pronounced in the FQ group compared to the NB and JZ groups, particularly with traits like the ChW, BL, HuW, and ChC exhibiting higher values in the DD genotype.Given that the FQ breed is more primitive and the NB and JZ breeds are more selectively bred, such differences might be attributed to breeding pressures.Notably, in the NB group, the InDels' effects on body height were particularly pronounced.In both InDels, the ID phenotypic values significantly surpassed those of DD, which could be due to heterosis.In summary, we hypothesize that the DD genotypes at the del72416 locus might confer higher feed conversion efficiency during Fuqing goat development.InDel variants can directly influence gene expression and function, including impacts on mRNA translation when located in exons [43] and transcription factor binding when occurring in introns [44][45][46].The presence of enhancer elements within introns and their potential regulatory impact on gene expression should particularly be taken into account [47,48].Given that a substantial number of InDels in gene intronic regions can significantly affect animal production traits, such as the notable 11 bp deletion within intron 9 of the CSN1S1 gene shown to influence milk yield and body measurements in goats [49,50], the newly identified InDels of the JAK2 gene could serve as valuable genetic markers for growth trait selection in goat breeding.

Conclusions
The del19008 and del72416 loci of the JAK2 gene were explored, and only two genotypes were designated in three goat breeds.The analyses revealed significant associations between these two loci and growth traits.The influence of the del72416 locus on growth traits was notably stronger in the FQ breed compared to the NB and JZ breeds.Additionally, in the NB population, the deleterious effects of the two InDels were primarily manifested in the BH.Moreover, the DD-ID diplotype was associated with inferior growth traits in the ChW and CaC in the FQ goats compared to the other diplotypes.In the NB population, the DD-DD diplotype exhibited a marked negative impact on the BH and HuWI, in contrast to the other diplotypes.Conversely, the DD-DD diplotype was associated with extremely significant superiority in the BLI and ChCI compared to the other diplotypes.These findings suggest that the two InDel polymorphisms significantly influence growth traits and could serve as valuable DNA markers for MAS in breeding programs aimed at enhancing growth characteristics in goats.

Figure 1 .
Figure 1.Electrophoresis diagrams and sequencing diagrams of the loci del19008 and Del72416 in the goat JAK2 gene.Note: M = molecular marker; del19008 genotypes (DD = 431 bp, ID = 431 bp and 436 bp): the area with the gray box is the sequence of the 5 bp deletion; del72416 genotypes (DD = 395 bp, ID = 395 bp and 405 bp): the region with the gray box is the bimodal sequence of the 10 bp deletion.

Figure 1 .
Figure 1.Electrophoresis diagrams and sequencing diagrams of the loci del19008 and Del72416 in the goat JAK2 gene.Note: M = molecular marker; del19008 genotypes (DD = 431 bp, ID = 431 bp and 436 bp): the area with the gray box is the sequence of the 5 bp deletion; del72416 genotypes (DD = 395 bp, ID = 395 bp and 405 bp): the region with the gray box is the bimodal sequence of the 10 bp deletion.

Figure 2 .
Figure 2. Phylogenetic analysis of JAK2 in 11 species.Note: The tree was constructed from the amino acid sequences via the neighbor-joining method using MEGA 5.1.The numbers on the joints are values from the bootstrap test, and the branch length represents the evolutionary time.

Figure 2 .
Figure 2. Phylogenetic analysis of JAK2 in 11 species.Note: The tree was constructed from the amino acid sequences via the neighbor-joining method using MEGA 5.1.The numbers on the joints are values from the bootstrap test, and the branch length represents the evolutionary time.

Table 2 .
Genotypic and allelic frequencies and genetic parameters of InDels within the JAK2 gene.

Table 2 .
Genotypic and allelic frequencies and genetic parameters of InDels within the JAK2 gene.

Table 3 .
Relationship between the InDels of the JAK2 gene and growth traits in goats.